GENERAL DISCUSSION

Physical, chemical and biological differences between conventional drug/gene and nanomedicine therapeutics.

As mentioned in previous sections, nanomaterials offer a number of advantages as delivery vectors. Some physical, chemical and biological differences between conventional drug/gene pharmaceuticals and nanomedicine therapeutics are highlighted in Table 4. In Paper III and Paper IV, it was clearly shown that the pDNA or siRNA per se would not be able to execute its effect without the delivery vectors. The Papers (I-IV) in this thesis aim to further investigate the safety and efficacy of nanomaterials as delivery vectors, as well as factors affecting their behaviors.

Table 4. Comparison between conventional drug/gene and nanomedicine therapeutics.

Characteristics Drugs Genes Nanomedicines

Synthesis Chemical synthesis Isolated from plant/animals or synthesized by means of genetic engineering

Formation of complexes between drugs/genes and nanovectors Molecular weight

or particle size

Low molecular weight, less than 1 nm

High molecular weight, usually a few nanometers

High molecular weight, usually around 1-100 nm Physical and

chemical characteristics

Characteristics of well-defined small molecular weight chemicals

Complex

physicochemical characteristics (e.g.

tertiary structure)

Characteristics of material science and particle science, including size, shape, mechanical properties, etc Interactions with

cells

Typically diffusion once inside the cell cytoplasm

Typically degraded by cellular enzymes

Typically confined intracellular location Interactions with

the human body

Poor

pharmacokinetics often lead to major side effects

Typically degraded by serum enzymes

Improved

pharmacokinetics

Physicochemical properties of nanomaterials in relation to their biocompatibility and gene delivery efficiency.

The work in this thesis emphasizes the basic understanding of the physicochemical properties of nanomaterials in relation to their biocompatibility and gene delivery efficiency. Although there is no clear consensus in the literature, some patterns are emerging. However, a larger sample size or meta-analysis would be necessary for deriving meaningful conclusions from statistical analyses of correlations between their physicochemical properties and biological endpoints. Moreover, the physicochemical properties of nanomaterials are interdependent (for example, synthesis of well-defined nanoparticles with different sizes also results in different surface charges) ¹²¹, therefore computer simulations would be needed to fully appreciate such complex relationships.

Chemical composition and crystallinity.

Currently, most nano-formulations that already exist on the market for in vivo delivery and imaging purposes are lipid and liposome based nanocomposites, polymers and iron oxide nanoparticles ¹. Indeed, chemical composition is among the determining factors for the biocompatibility of nanomaterials for biomedical applications. In Papers I-III, the use of silica nanomaterials as biocompatible nanomaterials for biomedical applications was investigated. In Paper II, amorphous silica nanoparticles were also compared to cerium oxide, titanium oxide, and zinc oxide nanoparticles of similar size.

Results from Paper II and others suggest that amorphous silica is considerably more biocompatible compared to many other materials such as zinc oxide, zirconia ¹²², etc. It is noteworthy that the crystalline form of silica is rather toxic and not suitable for biomedical applications ^{122, 123}. In Paper IV, the novel utilities of polythiophenes for gene delivery in biomedicine are explored. The toxicity of polythiophenes is not well understood, however, it was shown that polythiophene conductive polymers improve the biocompatibility of electrodes on primary mouse neurons ¹²⁴. Therefore, chemical composition and crystallinity has a strong impact on the biocompatibility of nanomaterials. Silica and polythiophene nanomaterials are potentially interesting materials for biomedical applications, with mesoporous silica nanoparticles entering the stage of preclinical development ¹²⁵. Other potential platforms include gold, magnetic nanoparticles, and carbon nanotubes ^{1, 18}.

Size.

There is substantial concern of a higher toxic potential at the nanolevel compared to the microlevel ¹²⁶, due to the higher proportion of atoms exposed at the surface of nanomaterials (compared to bulk materials of the same composition) as well as the ability of smaller particles to penetrate deeper into the body. In Paper I, the biocompatibility of silica nanomaterials with different size, surface charge, total surface area, hydrophobicity, and porosity were compared. These results, although inconclusive, suggest that smaller size particles seem to be more hemolytic and cytotoxic than larger ones at the same mass dose. Similarly, other studies found

size-dependent toxicity of amorphous silica particles in vitro and in vivo, with the smaller particles being more toxic. For example, smaller particles compared to larger ones were shown to be more cytotoxic in various cells by the MTT and LDH assays 121, 127-129, induce more apoptosis in human keratinocytes HaCaTa cells as detected by the annexin V-propidium iodide assay ¹³⁰, and induce more oxidative stress (ROS generation, lipid peroxidation and GSH depletion) in human hepatic L-02 cells ¹³¹. Mice intravenously injected with 75 nm silica particles induced liver injury at 30 mg/kg body weight, whereas 311 and 830 nm particles had no effect at 100 mg/kg ¹³². Feeding of mice for 10 weeks (total fed amount of 140 g/kg mice) with 30 nm silica nanoparticles induced higher levels of alanine aminotransferase (ALT) and fatty liver patterns compared to those of 30 µm silica microparticles (with similar liver retainment) ¹³³. Smaller polymer nanoparticles of 45 nm also showed higher cytotoxicity compared to larger 90 nm particles in terms of ROS production, adenosine-5'-triphosphate (ATP) depletion, tumor necrosis factor (TNF)-& release as well as the reduction of mitochondrial membrane potential in different cells ¹³⁴. Interestingly, it was reported that certain specific sizes can be substantially toxic, i.e. gold nanoclusters of 1.4 nm are remarkably more toxic than marginally smaller or larger gold nanoparticles potentially due to their interactions with the major grooves of DNA ¹³⁵.

Higher delivery efficiency in vivo is generally attributed to nanoparticles with a diameter around 100 nm, which are capable of circulating in the plasma for a few hours rather than seconds to minutes for smaller or larger particles ⁴. In addition to plasma circulation time that is a critical prerequisite for delivery, other factors such as cellular uptake are also important in governing the delivery efficiency of nanoparticle vectors.

Size-restrictions affect cellular uptake via different mechanisms of endocytosis (clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis, and clathrin/caveolin-independent endocytosis) ^{33, 105}. Nabiev et al. reported that the cell’s active transport machinery delivered nonfunctionalized nanocrystals to different regions of the cell in a size-specific manner ¹³⁶. He et al. showed that the availability of particles to be internalized is better for the smaller particles among particle sizes of 190, 420, and 1220 nm in various cells ¹²⁹. Lu et al. showed by confocal laser scanning microscopy and ICP-MS that cellular uptake in human cervical HeLa cells was optimal for silica particles of 50 nm compared to 30, 110, 170 and 280 nm ¹³⁷. Aoyama and co-workers demonstrated an optimal diameter around 50 nm for the cellular uptake of calix[4]-resorcarene-coated macrocyclic glycocluster amphiphiles or quantum dots ¹³⁸. Chan and co-workers also reported 40-50 nm diameter to be optimal for cellular internalization of pristine and protein-coated gold nanoparticles ^{139, 140}. Theoretical models converge on similar conclusions that particles ought to have a minimum diameter between 40 and 60 nm in order to achieve effective cellular uptake ¹⁴¹. Therefore, a delivery system has an optimal physical size in the nanometer range that facilitates their cellular binding and uptake (while also depending on other parameters), at least in non-phagocytic cells. On the other hand, it was suggested that larger particles are also able to enhance gene delivery in cell culture systems in vitro, which might be explained by the concentration of nucleic acids at the surface of cultured cells as a result of gravity ¹⁴².

Surface charge.

A positively charged surface is generally more toxic than a negatively charged surface, due to its potential interactions with many negatively charged biological molecules (such as glycolipids and nucleic acids) ¹⁴³. However, Slowing et al. showed that when amorphous silica particles were functionalized with carboxylic acid, their zeta-potential was similar (from -45.9 to -47.3 mV) but hemolysis was inhibited. This indicates that in the case of silica, hemolysis is specific to the silica surface despite the negative surface charge. The results in Paper I further points to the specific effects of surface silanol groups on the hemolytic and cytotoxic properties of silica particles. Isoda et al. found that intravenously administered amino group or carboxyl group modified silica nanoparticles were much less toxic than unmodified particles as shown by the level of liver injury (serum alanine aminotransferase level, liver hydroxyproline content, fibrosis) in mice ¹⁴⁴. These in vivo findings are also in line with the specific silica surface induced toxicity. For many other types of nanomaterials, such as polymers, higher positive charges are generally correlated with higher toxicity ^145-147.

Delivery vectors often carry positive charge to enable ionic complexation with nucleic acids. In Paper IV, it was demonstrated that the delivery efficiency of the cationic polythiophene was much higher than the zwitteronic polythiophenes. Cellular binding and uptake can be achieved either via non-specific adsorptive endocytosis (by providing excess positive surface charge) or specifically via receptor-mediated endocytosis ^{148, 149}. On the other hand, the strength of the ionic interactions between the delivery vectors and the nucleic acids can be a limiting factor later during the disassembly of the complexes ¹⁵⁰. In terms of in vivo delivery efficiency, the nanoparticle-nucleic acid complex is most desirable to be near neutral in order to avoid non-specific interactions with blood components, extracellular matrix and non-target cells or tissues in vivo.

Porosity.

Porosity may have an important role in determining the toxicity of nanoparticles.

Slowing et al. suggested that mesoporous silica particles have reduced hemolytic activity (compared to nonporous silica particles) which correlates to their lower external surface area as a result of their porous structures ³⁷. Similarly, lower hemolysis and cytotoxicity were generally observed for porous silica particles in Paper I, Paper III as well as a study by Rabolli et al. ¹²¹ in different cell types. However, more studies need to be performed to confirm this relationship.

Gao et al. demonstrated pore-size dependent drug release rate and therefore anticancer activity using mesoporous silica nanoparticles in drug sensitive and drug resistant MCF-7 cell lines ¹⁵¹. Na et al. showed pore-size dependent delivery of siRNA in vitro and in vivo using mesoporous silica nanoparticles, particles with larger pores (23 nm) being more efficient than those with smaller pores (2 nm) ¹⁵². In Paper III, nonporous

silica nanoparticles were shown to have superior delivery efficiency compared to mesoporous silica nanoparticles with pore diameters of 2.4 nm. Several reasons could account for this observation: these mesoporous silica nanoparticles with 2.4 nm pore diameter have small pore spaces that could not be efficiently explored for the accommodation of cargo; the different distribution of functional groups over the surface of mesoporous and nonporous silica particles may subsequently affect their binding to nucleic acids as well as aggregation state; there might be less cellular association of mesoporous compared to nonporous silica nanoparticles as shown in a quantitative study using ICP-MS ¹⁵³. Therefore, the dimensions of the pores could have a strong impact on the delivery efficiency of porous particles.

The effect of plasma/serum.

The effect of plasma/serum on nanoparticle behavior as well as their interactions with biological systems (particularly cytotoxicity and gene delivery efficiency) was examined in Papers I-III.

In Paper I, the presence of a biological corona over silica particles was confirmed by means of X-ray photon electron spectroscopy (XPS). In Paper I, it was demonstrated that the plasma/serum corona is primarily composed of proteins, but lipids may also be involved. The zeta-potential of plasma corona coated particles tends to be fairly similar (-20±5 mV) despite the very different zeta-potential of pristine particles (-10 to -50 mV). Monopoli et al. showed that the zeta-potential of 50 and 200 nm silica particles was modified by plasma corona (approx. from -25 to -10 mV), but the zeta-potential did not vary further with increasing concentrations of plasma (from 3% to 80%) ⁵⁶. In Paper II, it was shown that the serum corona reduced the aggregation of nanoparticles (SiO2, TiO2, CeO2, ZnO) and in some cases (e.g. ZnO) enhanced their dissolution.

Gualtieri et al. showed that 0.1% bovine serum albumin (BSA) reduced aggregation of silica nanoparticles ¹⁵⁴ whereas studies by Monopoli et al. and Drescher et al. observed higher aggregation of silica and polystyrene nanoparticles in the presence of plasma/serum ^{56, 155}. It was also shown that interactions of polymer-nucleic acid complexes with plasma proteins such as albumin leads to aggregation ^{156, 157}.

Interestingly, the coating of a pathogen with serum components is a mark for ingestion and destruction, a process termed opsonization, often resulting in phagocytosis and clearance from the circulation ¹⁵⁸. Similarly, plasma/serum protein coating over polymer nanoparticles accelerated their removal by phagocytic cells ^{157, 159}. Moreover, reduced cytotoxicity has been observed for nanoparticles in the presence of albumin ¹⁶⁰. It is however questionable whether the reduced toxicity is due to the antioxidant activities of albumin or the coating of albumin over the reactive surface of these nanoparticles. Indeed, it was shown in Paper I-III that the presence of plasma/serum abolished or delayed the toxicity of pristine silica nanoparticles, amino-functionalized silica nanoparticles and ZnO nanoparticles. In Paper I, further evidence was presented that the plasma corona coating of the silica surface protected silica nanoparticles against hemolysis and cytotoxicity. The human plasma/serum may thus serve the

function to mediate the in vivo distribution and excretion of nanoparticles and reduce their toxic effects in the systemic circulation.

On the other hand, reduced blood circulation time following the in vivo interactions of nanoparticle formulations with plasma proteins also impairs their delivery efficiency

157. Moreover, blood plasma/serum is also abundant in nucleic acid-degrading enzymes that can lead to a substantial loss of therapeutic effect ¹⁶¹. Therefore, research efforts are made towards using hydrophilic polymers (e.g. PEG) to shield nanoparticles from intensive interactions with blood proteins as well as searching for serum resistant formulations for delivery. For example, Lehto et al. showed that the delivery efficiency of a stearylated cell-penetrating peptide transportan 10 was maintained in the presence of serum proteins mimicking in vivo conditions ¹⁶². Silica particles provide promising serum resistant features for in vivo applications ¹⁵², although some discrepancy exists in our study and the literature. In Paper III, amino-functionalized silica particles displayed higher delivery efficiency for pDNA in MCF-7 cells in the presence of 10% serum than in the absence of serum, whereas Na et al. observed marginally lower delivery efficiency for siRNA in human cervical carcinoma HeLa cells with 10% serum than without serum ¹⁵². Nevertheless, Xiao et al. confirmed the protection of DNA by mesoporous silica particles from serum nucleases ¹⁶³.

In vitro vs. in vivo.

Although numerous studies have used cell models to investigate the biocompatibility of nanomaterials and their applications for gene delivery, it is questionable how much knowledge from in vitro studies can be readily transferred to in vivo situations ¹⁶⁴. First, in vitro systems lack the complexities of in vivo pharmacokinetics, physiological structures, and systemic responses. Second, particles, unlike small molecules, do not necessarily evenly distribute in fluids. On the contrary, they may exhibit distinct behaviors in body fluids and cell cultures ¹⁴². Third, cellular phenotypes (such as their repertoire of expressed receptors) may show significant variations in in vitro cell cultures ¹⁶⁵. Nevertheless, in vitro studies may still prove to be useful in nanomedical research for identifying similar patterns of biologic activity and understanding the mechanisms of action ¹⁶⁶.

In addition to the in vivo approach for the administration of therapeutic nucleic acid formulations, the ex vivo approach first delivers the genetic material into cells grown in vitro (usually autologous cells from the same patient) and then introduce those transfected cells into the patient ¹⁶⁷.